Standardized experiments in mutant mice reveal behavioural similarity on 129S5 and C57BL/6J backgrounds.

Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non-standardized behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/6J backgrounds generated in a standardized battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently used group sizes, a single retest of a significant result conforming to the commonly used P < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P-value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies.

Human cognitive ability is influenced by genetic variation in components of postsynaptic signalling complexes assembled by NMDA receptors and MAGUK proteins.

Differences in general cognitive ability (intelligence) account for approximately half of the variation in any large battery of cognitive tests and are predictive of important life events including health. Genome-wide analyses of common single-nucleotide polymorphisms indicate that they jointly tag between a quarter and a half of the variance in intelligence. However, no single polymorphism has been reliably associated with variation in intelligence. It remains possible that these many small effects might be aggregated in networks of functionally linked genes. Here, we tested a network of 1461 genes in the postsynaptic density and associated complexes for an enriched association with intelligence. These were ascertained in 3511 individuals (the Cognitive Ageing Genetics in England and Scotland (CAGES) consortium) phenotyped for general cognitive ability, fluid cognitive ability, crystallised cognitive ability, memory and speed of processing. By analysing the results of a genome wide association study (GWAS) using Gene Set Enrichment Analysis, a significant enrichment was found for fluid cognitive ability for the proteins found in the complexes of N-methyl-D-aspartate receptor complex; P=0.002. Replication was sought in two additional cohorts (N=670 and 2062). A meta-analytic P-value of 0.003 was found when these were combined with the CAGES consortium. The results suggest that genetic variation in the macromolecular machines formed by membrane-associated guanylate kinase (MAGUK) scaffold proteins and their interaction partners contributes to variation in intelligence.

Deriving structural and functional insights from a ligand-based hierarchical classification of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute the largest known family of cell-surface receptors. With hundreds of members populating the rhodopsin-like GPCR superfamily and many more awaiting discovery in the human genome, they are of interest to the pharmaceutical industry because of the opportunities they afford for yielding potentially lucrative drug targets. Typical sequence analysis strategies for identifying novel GPCRs tend to involve similarity searches using standard primary database search tools. This will reveal the most similar sequence, generally without offering any insight into its family or superfamily relationships. Conversely, searches of most 'pattern' or family databases are likely to identify the superfamily, but not the closest matching subtype. Here we describe a diagnostic resource that allows identification of GPCRs in a hierarchical fashion, based principally upon their ligand preference. This resource forms part of the PRINTS database, which now houses approximately 250 GPCR-specific fingerprints (http://www.bioinf.man.ac.uk/dbbrowser/gpcrPRINTS/). This collection of fingerprints is able to provide more sensitive diagnostic opportunities than have been realized by related approaches and is currently the only diagnostic tool for assigning GPCR subtypes. Mapping such fingerprints on to three-dimensional GPCR models offers powerful insights into the structural and functional determinants of subtype specificity.

Prediction of the coupling specificity of G protein coupled receptors to their G proteins.

UNLABELLED: G protein coupled receptors (GPCRs) are found in great numbers in most eukaryotic genomes. They are responsible for sensing a staggering variety of structurally diverse ligands, with their activation resulting in the initiation of a variety of cellular signalling cascades. The physiological response that is observed following receptor activation is governed by the guanine nucleotide-binding proteins (G proteins) to which a particular receptor chooses to couple. Previous investigations have demonstrated that the specificity of the receptor-G protein interaction is governed by the intracellular domains of the receptor. Despite many studies it has proven very difficult to predict de novo, from the receptor sequence alone, the G proteins to which a GPCR is most likely to couple. We have used a data-mining approach, combining pattern discovery with membrane topology prediction, to find patterns of amino acid residues in the intracellular domains of GPCR sequences that are specific for coupling to a particular functional class of G proteins. A prediction system was then built, being based upon these discovered patterns. We can report this approach was successful in the prediction of G protein coupling specificity of unknown sequences. Such predictions should be of great use in providing in silico characterisation of newly cloned receptor sequences and for improving the annotation of GPCRs stored in protein sequence databases. AVAILABILITY: http://www.ebi.ac.uk/~croning/coupling.html.

Evaluation of methods for the prediction of membrane spanning regions.

MOTIVATION: A variety of tools are available to predict the topology of transmembrane proteins. To date no independent evaluation of the performance of these tools has been published. A better understanding of the strengths and weaknesses of the different tools would guide both the biologist and the bioinformatician to make better predictions of membrane protein topology. RESULTS: Here we present an evaluation of the performance of the currently best known and most widely used methods for the prediction of transmembrane regions in proteins. Our results show that TMHMM is currently the best performing transmembrane prediction program.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

InterPro--an integrated documentation resource for protein families, domains and functional sites.

MOTIVATION: InterPro is a new integrated documentation resource for protein families, domains and functional sites, developed initially as a means of rationalising the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. RESULTS: Merged annotations from PRINTS, PROSITE and Pfam form the InterPro core. Each combined InterPro entry includes functional descriptions and literature references, and links are made back to the relevant parent database(s), allowing users to see at a glance whether a particular family or domain has associated patterns, profiles, fingerprints, etc. Merged and individual entries (i.e. those that have no counterpart in the companion resources) are assigned unique accession numbers. Release 1.2 of InterPro (June 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification (PTMs) encoded by 6581 different regular expressions, profiles, fingerprints and Hidden Markov Models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1000000 hits from 264333 different proteins out of 384572 in SWISS-PROT and TrEMBL).

PRINTS-S: the database formerly known as PRINTS.

The PRINTS database houses a collection of protein family fingerprints. These are groups of motifs that together are diagnostically more potent than single motifs by virtue of the biological context afforded by matching motif neighbours. Around 1200 fingerprints have now been created and stored in the database. The September 1999 release (version 24.0) encodes approximately 7200 motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. In addition to its continued steady growth, we report here several major changes to the resource, including the design of an automated strategy for database maintenance, and implementation of an object-relational schema for more efficient data management. The database is accessible for BLAST, fingerprint and text searches at http://www.bioinf.man.ac. uk/dbbrowser/PRINTS/

Sodium homeostasis in rat hippocampal slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels.

The breakdown in brain ionic homeostasis during energy deprivation (anoxic depolarization [AD]) is intimately linked to neuronal injury. We studied the role of one particular route of Na+ influx, voltage-sensitive Na+ channels, in the AD induced by O2 and/or glucose deprivation. We recorded extracellular Na+ concentration ([Na+]e) and direct current potential (DCP) in the CA1 stratum pyramidale of hippocampal slices using Na+-selective microelectrodes. Tetrodotoxin (0.1-1 microM) delayed the occurrence of AD and reduced the peak change in both [Na+]e and DCP during AD. However the tetrodotoxin effects were overcome by a concomitant reduction in extracellular glucose during anoxia. We conclude that: (1) the activation of voltage-gated Na+ channels is involved in the triggering of AD; (2) there may be a critical level of energy depletion when AD occurs and different mechanisms may underlie AD during hypoxia, compared to O2 and glucose deprivation.

Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro.

We have systematically compared anoxia-induced effects in the CA1 stratum pyramidale of rat hippocampal slices maintained in Haas-type interface, and fully-submerged recording chambers to determine whether heterogeneous responses occur in response to oxygen deprivation, between these brain slice chambers. Extracellular K+ concentration ([K+]e), extracellular d.c. potential, and synaptically-evoked field potentials were measured using a K+-selective microelectrode. In slices maintained in the interface chamber, anoxia resulted in a dramatic disturbance of K homeostasis; during the first approximately 3 min of anoxia, [K+]e increased to ca. 5 mM and synaptic transmission was abolished. Thereafter, [K+]e increased explosively to ca. 35 mM, and a negative shift in the extracellular d.c. potential occurred. In slices maintained fully-submerged, even prolonged anoxia (60 min) caused only a very modest increase in [K+]e to ca. 5 mM and a negative shift in the extracellular potential never occurred. However, when glucose deprivation was combined with anoxia, both a dramatic increase in [K+]e and a negative shift in the d.c. potential were observed. We conclude that major differences exist in the degree of disruption of ionic homeostasis by anoxia in rat hippocampal slices when they are maintained at a gas-liquid interface, compared to when they are completely immersed. Possible factors underlying this difference include heterogeneous rates of glucose diffusion into the tissue slices, and washout from the extracellular space of accumulating substances such as K+ by the perfusing artificial cerebrospinal fluid (ACSF). The changes observed in the interface chamber may more closely replicate the dramatic disturbance of K+ homeostasis that is commonly observed in rodent brain in response to anoxia in vivo.

Action of adenosine receptor antagonists on hypoxia-induced effects in the rat hippocampus in vitro.

1. We have studied three hypoxia-induced phenomena in the CA1 stratum pyramidale of the rat hippocampal slice: (a) the increase in extracellular potassium ion concentration ([K+]e) measured with ion-sensitive microelectrodes, (b) the intracellularly-recorded pyramidal cell hyperpolarization and (c) the extracellularly-recorded depression of the synaptically-evoked field potential recorded in stratum pyramidale. 2. The extracellular potassium ion concentration ([K+]e) rose from 3 mM to 4.1-4.4 mM at a time when the pyramidal cells hyperpolarized by about 6 mV and neurotransmission was virtually abolished. 3. Presumed glial cells depolarized in response to hypoxia. The shape and time course of this response was remarkably similar to the rise in [K+]e so induced. This is consistent with findings that glial cell membrane potential is dependent on transmembrane K+ gradient. 4. We investigated the effects of theophylline (100 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM) on these effects. We have found that these compounds attenuated by about half the hypoxia-induced increase in [K+]e; however, they did not reduce the hypoxia-induced hyperpolarization. We have confirmed that they dramatically reduced the suppression of excitatory transmission caused by the hypoxia. We conclude that adenosine A1 receptors may be involved in the alteration of K+ homeostasis in the hippocampal slice during hypoxia.